Lab 5: Determination of
Antimicrobial Effects of Microbial Exracts
INTRODUCTION
There are a few groups
of bacteria that can produce antimicrobial substances. An antimicrobial is an
agent that kills microorganism or inhibits their growth. Antimicrobial
medicines can be grouped according to the microorganisms they act primarily
against. For example, antibacterials are used against bacteria and antifungals
are used against fungi. They can also be classified according to their
function. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are
included among these antimicrobial compounds. Since nowadays consumers demand
“natural” and minimally processed” food therefore there is a rising of interest
on naturally produced antimicrobial agents like bacteriocins.
Bacteriocins are proteins or complexed
proteins biologically active with antimicrobial action against other bacteria,
principally closely related species. They are produced by bacteria and are
normally not termed antibiotics in order to avoid confusion and concern with
therapeutic antibiotics, which can potentially illicit allergic reactions in
humans and other medical problems
Bacteriocin production could be
considered as an advantage for food and feed producers since, in sufficient
amounts, these peptides can kill or inhibit pathogenic bacteria that compete
for the same ecological niche or nutrient pool. This role is supported by the
fact that many bacteriocins have a narrow host range, and is likely to be most
effective against related bacteria with nutritive demands for the same scarce
resources
Lactic acid bacteria (LAB) are
characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant,
able to ferment carbohydrates for energy and lactic acid production. LAB
bacteriocins can work via different mechanisms to exert an antimicrobial
effect, but the cell envelope is generally the target. The initial
electrostatic attraction between the target cell membrane and the bacteriocin
peptide is thought to be the driving force for subsequent events.
Materials and Reagents
MRS
broth
Sterile
filter paper disk
Forceps
Sterile
universal bottles
Cultures
of LAB and spoilage/pathogenic organisms
Bench-top
refrigerated centrifuge
Incubator
30oc and 37oc
UV/V
is spectrophotometer
Distilled
deionized water
Trypticase
soy agar
Brain
heart infusion agar
Yeast
extract
PROCEDURE
Part
1: Determination Of Bacteriocin Activity Via Agar Diffusion Test
1. All the petri dishes
are labelled according to the spoilage organisms and strains on LAB used.
2. Each plate was only
used for one strain of spoilage organism and one strain of LAB. Plate was
divided into 2 parts, each for on replicate.
3. Each group has 1
strain of LAB and 1 strain of spoilage/pathogenic organism.
4.10ml of trypticase
soy-yeast extract agar (TSAYE) was loaded into the labeled petri ish and the
agar was ensured to fully cover the entire surface of the plate. It is waited
until it solidifies.
5. 2ml of broth
containing the spoilage organism was innoculated into 10ml of brain hear
infusion (BHI) agar and vortex.
6. The mixture was
loaded on top of the TSAYE agar layer and ensurd that it covered the entire
surface and waited to solidify.
7. The broth containing
the lab cultures was centrifuge. The supernatent was used as a extracellular
extracts.
8. A sterile filter
paper disk is picked up aseptically with a sterile forcep and a disk is dipped
into the extracellular extract.
9. The paper disk was
placed on top of the solidifies BHI agar.
10. The plates were
inoculated for 24-28 hours at 37oC.
11. Upon incubation,
the inhibition zones were measured (in cm) and is recorded.
Part
2: Determination Of Bacteriocin Activity Via Optical Density
1. The broth was
containing LAB cultures were centrifuge. The supernatent is used as
extracellular extracts.
2. Each group has 1
strain of LAB and 1 strain of spoilage/pathogenic organism.
3. 5 ml of
double-strength MRS was added with 1 ml of cultures containing spoilage/
pathogenic bacteria and the mixture was vortex.
4. A serial dilution of
the extracellular extracts (diluted 0x, 2x, 10x, 50x, 100x) were prepared.
5. 5 ml of each extracellular
extracts dilution was added into mixture as prepared in step(3).
6. The mixtures were
incubated for 12-15 hours at 37ºC.
7. A control using 5 ml
of double-strength MRS, 1 ml of cultures containing spoilage/ pathogenic
bacteria, and 5 ml of sterile peptone was prepared. The mixtures were incubated
for 12-15 hours at 37ºC.
8. A negative control
for ‘auto zero’ via the spectrophotometer was prepared. 5 ml of double-strength
MRS was added with 2 ml of distilled water. (Need not to be incubated)
9. Upon incubation, the
optical density of the spoilage/ pathogenic bacteria at 600 nm was measured.
The same was performed for the control as well.
10. One arbitrary unit
(AU) is defined as the dilution factor of the extracellular extract that
inhibited 50% of the spoilage/ pathogenic bacteria growth and expressed as
AU/ml.
11. 50% of the
spoilage/ pathogenic bacteria growth were determined from the OD600 of the
control.
RESULTS
Part
1: Determination Of Bacteriocin Activity Via Agar Diffusion Test
Presence of inhibition zone
Absence of inhibition zone
Strain
of lab
|
Strain
of spoilage/pathogenic bacteria
|
Name
|
Inhibition
Zone (cm)
|
Average
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 1
|
1
and 1.1
|
1.05
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 2
|
1
and 0.9
|
0.9
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 3
|
0.6
and 0.7
|
0.65
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 4
|
0.7
and 0.7
|
0.7
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 5
|
No
inhibition zone
|
|
Lactobacillus
fermentum
|
S.Aureus
|
sample 6
|
No
inhibition zone
|
Part 2: Determination of Bacteriocin Activity Via Optical Density
Dilution
|
OD600
of Spoilage/ Pathogenic Bacteria
|
0x
|
0.269
|
2x
|
0.397
|
10x
|
0.448
|
50x
|
0.174
|
100x
|
0.123
|
Equation
|
y=
-0.0026x + 0.3678
|
OD600
of Contol
| 0.107 |
50%
of OD600
|
0.0535
|
AU/ml
|
100/11= 9.091
|
DISCUSSION
Part
1: Determination Of Bacteriocin Activity Via Agar Diffusion Test
1. Bacteriocins
are bactericidal, antibiotic-like substances, apparently protein in nature,
which are produced by many bacteria and have a killing action on strains
of the same or closely related species.
2.The larger the
inhibition zone(no bacteria growing area) on the agar medium,means that the
bacteriocin is effective on the pathogenic bacteria and vise versa.
3. Type of the LAB of
bacteriocin that being used is Lactobacillus fermentum and the bacteria
that we used is S.Aureus.
4.The bacteriocin will
cause the destruction of the membrane potential by forming the pores on the
pathogenic bacteria. It will cause the destruction of the membrane
potential by forming the pores on the pathogenic bacteria.It will inhibits the
nucleolytic activity of the pathogenic bacteria strains which breaks down the
DNA chromosomes as well as RNA.Then the bacteriocin will inhibits the protein
synthesis of the pathogenic bacterias but does not kills them.
5.As for the none
inhibition zone exist result are because not enough Lactobacillus fermentum are
being aplied around the pathogenic bacteria.This is because without
adequate numbers of Lactobacillus fermentum, the point of critical mass which
is needed cannot occur and the bacteria will be unable to have the desired
impact on the symptoms being treated.
Part 2: Determination Of Bacteriocin Activity Via Optical Density
1.Optical density,
measured in a spectrophotometer, can be used as a measure of the concentration
of bacteria in a suspension. As visible light passes through a cell suspension
the light is scattered. Greater scatter indicates that more bacteria or other
material is present. The amount of light scatter can be measured in a
spectrophotometer. Typically, when working with a particular type of cell, you
would determine the optical density at a particular wavelength that correlates
with the different phases of bacterial growth. Generally we will want to use
cells that are in their mid-log phase of growth. Typically the OD600 is
measured.
2. One arbitrary (AU)
is defined as the dilution factor of the extracellular extract that inhibited
50% of the spoilage/pathogenic bacteria
growth and expressed as AU/mL.
Control : Abs600 = Z.
Thus, 50% of Z = Z/2
Y= mx + c ; Thus x=
(y-c)/m
When y= Z/2, thus x=
(Z/2-c)/m
3. As the serial of
dilution increse, the optical density will increase to indicate that the Lab
which is Lactobacillus Plantarum has strong microbial effec on the pathogenic
bacteria which is E.coli.
4. The control value of
the experiment is 0.209 and the value of the absorbant is 0.102.
5. From the result,the
graph shows that as the serial dilution increases, the optical density
decreases. This shows that there is negative inhibition of the pathogenic
bacteria. This might be caused by the using of distilled water during the
process of serial dilution. As we know, the distilled water is colourless. When
the Lactobacillus Plantarum is diluted with distilled water,
the optical colour density will become very much lower compared to the normal
colour density of Lactobacillus Plantarum culture. So, the
results obtained is wrong.
6. Therefore, the
peptone is suggested to replace with the distilled water in the serial dilution
of Lactobacillus Plantarum culture as the colour of
peptone is quite similar with the culture.
CONCLUSION
LAB is a useful
bacteria used to produce bacteriocin that can inhibit the growth of
bacteria.The use of strains that produce multiple bacteriocins could be
advantageous to limit the potential emergence of bacteriocin-resistant
populations An important aspect to take into consideration in relation to the
commercial use of bacteriocins is the tolerance or resistance of certain
pathogenic bacterial species that are normally sensitive since it may
compromise the antibacterial efficiency of these compounds
REFERENCES
http://www.academia.edu/3745035/Bacteriocins_Nature_Function_and_Structure
http://www.scielo.br/scielo.php?pid=s1516-89132007000300018&script=sci_arttext
http://nootriment.com/lactobacillus-fermentum/
http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html
